Compositions comprising bacterial strains

ABSTRACT

The invention provides compositions comprising bacterial strains for treating and preventing brain injury.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.16/691,169, filed Nov. 21, 2019, which is a continuation ofInternational Application No. PCT/GB2018/051386, filed May 22, 2018,which claims the benefit of Great Britain Application No. 1708176.1,filed May 22, 2017, Great Britain Application No. 1714305.8, filed Sep.6, 2017, Great Britain Application No. 1714309.0, filed Sep. 6, 2017,Great Britain Application No. 1714298.5, filed Sep. 6, 2017, GreatBritain Application No. 1716493.0, filed Oct. 9, 2017, and Great BritainApplication No. 1718551.3, filed Nov. 9, 2017, all of which are herebyincorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ANSI format and is hereby incorporated byreference in its entirety. Said ANSI copy, created on Jul. 16, 2021, isnamed 56708 724 302 SL and is 12,288 bytes in size.

TECHNICAL FIELD

This invention is in the field of compositions comprising bacterialstrains isolated from the mammalian digestive tract and the use of suchcompositions in the treatment of disease.

BACKGROUND TO THE INVENTION

The human intestine is thought to be sterile in utero, but it is exposedto a large variety of maternal and environmental microbes immediatelyafter birth. Thereafter, a dynamic period of microbial colonization andsuccession occurs, which is influenced by factors such as delivery mode,environment, diet and host genotype, all of which impact upon thecomposition of the gut microbiota, particularly during early life.Subsequently, the microbiota stabilizes and becomes adult-like [1]. Thehuman gut microbiota contains more than 1500 different phylotypes,dominated in abundance levels by two major bacterial divisions (phyla),the Bacteroidetes and the Firmicutes [2-3]. The successful symbioticrelationships arising from bacterial colonization of the human gut haveyielded a wide variety of metabolic, structural, protective and otherbeneficial functions. The enhanced metabolic activities of the colonizedgut ensure that otherwise indigestible dietary components are degradedwith release of by-products providing an important nutrient source forthe host and additional health benefits. Similarly, the immunologicalimportance of the gut microbiota is well-recognized and is exemplifiedin germfree animals which have an impaired immune system that isfunctionally reconstituted following the introduction of commensalbacteria [4-6].

Dramatic changes in microbiota composition have been documented ingastrointestinal disorders such as inflammatory bowel disease (IBD). Forexample, the levels of Clostridium cluster XIVa bacteria and Clostridiumcluster XI (F. prausnitzii) are reduced in IBD patients whilst numbersof E. coli are increased, suggesting a shift in the balance of symbiontsand pathobionts within the gut [7-11].

In recognition of the potential positive effect that certain bacterialstrains may have on the animal gut, various strains have been proposedfor use in the treatment of various diseases (see, for example,[12-15]). Human gut bacteria are known to produce short chain fattyacids (SCFAs) which have been proposed to treat autoimmune and/orinflammatory disorders [16]. A number of strains, including mostlyLactobacillus and Bifidobacterium strains, have been proposed for use intreating various bowel disorders (see [17] for a review). Bacterialstrains, in particular butyrogenic bacteria, have been proposed to treatand/or prevent Clostridium difficile infection [18].

Clostridium butyricum has been shown to attenuate cerebralischemia/reperfusion in mice models ([19] and [20]). Administration ofC. butyricum led to the modulation of the gut microbiota, whichcorrelated with changes in the levels of various neurotrophins andmonoamine transmitters that are involved in brain function. Due to thecomplexity of molecules involved, the authors conclude that themechanism by which C. butyricum produced these neuroprotective effectswas impossible to identify. Treatment with C. butyricum led to areduction in apoptosis via decreasing the levels of capase-3 in responseto PI3K/Akt activation. The PI3K/Akt pathway has been widely reported toparticipate in protection against psychiatric disorders.

Strains of the genus Blautia have also been proposed for use inmodulating the microbial balance of the digestive ecosystem [21] andparticular species have been proposed for use in treating systemicdiseases distanced from the gut [22]. However, the relationship betweendifferent bacterial strains and different diseases, and the preciseeffects of particular bacterial strains on the gut and at a systemiclevel and on any particular types of diseases, are poorly characterised.

There is a requirement for the potential effects of gut bacteria to becharacterised so that new therapies using gut bacteria can be developed.

SUMMARY OF THE INVENTION

The inventors have developed new therapies for treating brain injury. Inparticular, the inventors have identified that bacterial strains fromthe genus Blautia can be effective for treating brain injury. Asdescribed in the examples, oral administration of compositionscomprising Blautia hydrogenotrophica may treat brain injury, aidingrecovery and improving neurological function, motor abilities and socialrecognition in a mouse occlusion model of brain injury in patients.Therefore, in a first embodiment, the invention provides a compositioncomprising a bacterial strain of the genus Blautia, for use in a methodof treating brain injury.

In preferred embodiments, the invention provides a compositioncomprising a bacterial strain of the genus Blautia, for use in a methodof treating or preventing brain injury. In preferred embodiments, thecompositions of the invention are for use in treating stroke. Inparticularly preferred embodiments, the stroke is cerebral ischemia. Insome embodiments the cerebral ischemia is ischemic stroke. In someembodiments, the stroke is hemorrhagic stroke.

In preferred embodiments of the invention, the bacterial strain in thecomposition is of Blautia hydrogenotrophica. Closely related strains mayalso be used, such as bacterial strains that have a 16s rRNA sequencethat is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical tothe 16s rRNA sequence of a bacterial strain of Blautiahydrogenotrophica. Preferably, the bacterial strain has a 16s rRNAsequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%identical to SEQ ID NO:5. Most preferably, the bacterial strain in thecomposition is the Blautia hydrogenotrophica strain deposited underaccession number DSM 14294.

In further embodiments of the invention, the bacterial strain in thecomposition is of Blautia stercoris. Closely related strains may also beused, such as bacterial strains that have a 16s rRNA sequence that is atleast 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16s rRNAsequence of a bacterial strain of Blautia stercoris. Preferably, thebacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%,98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:1 or 3. Preferably, thesequence identity is to SEQ ID NO:3. Preferably, the bacterial strainfor use in the invention has the 16s rRNA sequence represented by SEQ IDNO:3.

In further embodiments of the invention, the bacterial strain in thecomposition is of Blautia wexlerae. Closely related strains may also beused, such as bacterial strains that have a 16s rRNA sequence that is atleast 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16s rRNAsequence of a bacterial strain of Blautia wexlerae. Preferably, thebacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%,98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:2 or 4. Preferably, thesequence identity is to SEQ ID NO:4. Preferably, the bacterial strainfor use in the invention has the 16s rRNA sequence represented by SEQ IDNO:4.

In certain embodiments, the composition of the invention is for oraladministration. Oral administration of the strains of the invention canbe effective for treating brain injury. Also, oral administration isconvenient for patients and practitioners and allows delivery to and/orpartial or total colonisation of the intestine.

In certain embodiments, the composition of the invention comprises oneor more pharmaceutically acceptable excipients or carriers.

In certain embodiments, the composition of the invention comprises abacterial strain that has been lyophilised. Lyophilisation is aneffective and convenient technique for preparing stable compositionsthat allow delivery of bacteria.

In certain embodiments, the invention provides a food product comprisingthe composition as described above.

In certain embodiments, the invention provides a vaccine compositioncomprising the composition as described above.

Additionally, the invention provides a method of treating brain injury,comprising administering a composition comprising a bacterial strain ofthe genus Blautia.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: Analysis of neurological function and motor abilities in acerebral ischemia-reperfusion I/R mouse model using the Inverted Screentest, in mice with or without treatment with Blautia hydrogenotrophica.

FIG. 2: Analysis of neurological function and social recognition in acerebral ischemia-reperfusion I/R mouse model using the SocialRecognition test, in mice with or without treatment with Blautiahydrogenotrophica.

FIG. 3: Effect of Blautia hydrogenotrophica (10¹⁰/day for 14 days) onshort chain fatty acids production (RMN ¹H) in caecal contents ofhealthy HIM rats.

FIG. 4: qPCR evaluation of B. hydrogenotrophica population in faecalsamples of IBS-HMA rats treated or not with a composition comprising B.hydrogenotrophica (BlautiX) for 28 days.

FIGS. 5A and 5B: Short chain fatty acids (SCFA) concentrations in caecalsamples of IBS-HMA rats treated or not with B. hydrogenotrophica(Blautix) for 28 days. FIG. 5A shows concentration of total SCFA. FIG.5B shows concentration of Acetic acid, Propionic acid and Butyric acid.

FIGS. 6A, 6B, and 6C: Histological evaluation of tissue samples frommice treated with bacterial composition comprising B. hydrogenotrophica(Blautix). FIG. 6A shows the semi-quantitative pyknosis score ofhistology H&E stained tissue samples. Mice administered with thecomposition of the invention (5M) display significantly reduced pyknosisscore compared to the negative control mice (2M) (p<0.0001). FIG. 6Bshows the semi-quantitative vacuolisation score of histology H&Estrained tissue samples. Mice administered with the composition of theinvention (5M) display significantly reduced vacuolisation scorecompared to the negative control (2M) (p=0.098). FIG. 6C showsrepresentative photographs of H&E stained slides—dark pyknotic neuronsnuclei and some swollen pale vacuoles in some affected neuronscytoplasm.

DISCLOSURE OF THE INVENTION Bacterial Strains

The compositions of the invention comprise a bacterial strain of thegenus Blautia. The examples demonstrate that bacteria of this genus areuseful for treating brain injury. The preferred bacterial strains are ofthe species Blautia hydrogenotrophica, Blautia stercoris and Blautiawexlerae. Other preferred bacterial strains for use in the invention areBlautia producta, Blautia coccoides and Blautia hansenii.

Examples of Blautia strains for use in the invention include Blautiahydrogenotrophica, B. stercoris, B. faecis, B. coccoides, B. glucerasea,B. hansenii, B. luti, B. producta, B. schinkii and B. wexlerae. TheBlautia species are Gram-reaction-positive, non-motile bacteria that maybe either coccoid or oval and all are obligate anaerobes that produceacetic acid as the major end product of glucose fermentation [23].Blautia may be isolated from the human gut, although B. producta wasisolated from a septicaemia sample.

Blautia hydrogenotrophica (previously known as Ruminococcushydrogenotrophicus) has been isolated from the guts of mammals, isstrictly anaerobic, and metabolises H₂/CO₂ to acetate, which may beimportant for human nutrition. The type strain of Blautiahydrogenotrophica is S5a33=DSM 14294=JCM 14656. The GenBank accessionnumber for the 16S rRNA gene sequence of Blautia hydrogenotrophicastrain S5a36 is X95624.1 (disclosed herein as SEQ ID NO:5). Thisexemplary Blautia hydrogenotrophica strain is described in [19] and[24]. The S5a33 strain and the S5a36 strain correspond to two subclonesof a strain isolated from a faecal sample of a healthy subject. Theyshow identical morphology, physiology and metabolism and have identical16S rRNA sequences. Thus, in some embodiments, the Blautiahydrogenotrophica for use in the invention has the 16S rRNA sequence ofSEQ ID NO:5.

The Blautia hydrogenotrophica bacterium deposited under accession numberDSM 14294 was tested in the examples and is also referred to herein asstrain BH and Blautix. Strain BH is the preferred strain of theinvention. Strain BH was deposited with the Deutsche Sammlung vonMikroorganismen [German Microorganism Collection] (Mascheroder Weg 1b,38124 Braunschweig, Germany) under accession number DSM 14294 as “S5a33”on 10 May 2001. The depositor was INRA Laboratoire de Microbiologie CRde Clermont-Ferrand/Theix 63122 Saint Genes Champanelle, France.Ownership of the deposits has passed to 4D Pharma Plc by way ofassignment. 4D Pharma Plc has authorised, by way of an agreement, 4DPharma Research Limited to refer to the deposited biological material inthe application and has given its unreserved and irrevocable consent tothe deposited material being made available to the public.

The GenBank accession number for the 16S rRNA gene sequence of Blautiastercoris strain GAM6-1^(T) is HM626177 (disclosed herein as SEQ IDNO:1). An exemplary Blautia stercoris strain is described in [25]. Thetype strain of Blautia wexlerae is WAL 14507=ATCC BAA-1564=DSM 19850[19]. The GenBank accession number for the 16S rRNA gene sequence ofBlautia wexlerae strain WAL 14507 T is EF036467 (disclosed herein as SEQID NO:2). This exemplary Blautia wexlerae strain is described in [19].

All microorganism deposits were made under the terms of the BudapestTreaty and thus viability of the deposit is assured. Maintenance of aviable culture is assured for 30 years from the date of deposit. Duringthe pendency of the application, access to the deposit will be affordedto one determined by the Commissioner of the United States Patent andTrademark Office to be entitled thereto. All restrictions on theavailability to the public of the deposited microorganisms will beirrevocably removed upon the granting of a patent for this application.The deposit will be maintained for a term of at least thirty (30) yearsfrom the date of the deposit or for the enforceable life of the patentor for a period of at least five (5) years after the most recent requestfor the furnishing of a sample of the deposited material, whichever islongest. The deposit will be replaced should it become necessary due toinviability, contamination or loss of capability to function in themanner described in the specification.

A preferred Blautia stercoris strain is the strain deposited underaccession number NCIMB 42381, which is also referred to herein as strain830. A 16S rRNA sequence for the 830 strain is provided in SEQ ID NO:3.Strain 830 was deposited with the international depositary authorityNCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) by GTBiologics Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS,Scotland) on 12th March 2015 as “Blautia stercoris 830” and was assignedaccession number NCIMB 42381. GT Biologics Ltd. subsequently changed itsname to 4D Pharma Research Limited.

A preferred Blautia wexlerae strain is the strain deposited underaccession number NCIMB 42486, which is also referred to herein as strainMRX008. A 16S rRNA sequence for the MRX008 strain is provided in SEQ IDNO:4. Strain MRX008 was deposited with the international depositaryauthority NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland)by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen,AB25 2ZS, Scotland) on 16 Nov. 2015 as “Blaqutia/Ruminococcus MRx0008”and was assigned accession number NCIMB 42486.

Bacterial strains closely related to the strain tested in the examplesare also expected to be effective for treating brain injury. In certainembodiments, the bacterial strain for use in the invention has a 16srRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%identical to the 16s rRNA sequence of a bacterial strain of Blautiahydrogenotrophica. Preferably, the bacterial strain for use in theinvention has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%,99%, 99.5% or 99.9% identical to SEQ ID NO:5.

In certain embodiments, the bacterial strain for use in the inventionhas a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%or 99.9% identical to the 16s rRNA sequence of a bacterial strain ofBlautia stercoris. Preferably, the bacterial strain for use in theinvention has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%,99%, 99.5% or 99.9% identical to SEQ ID NO:1 or SEQ ID NO:3. Preferably,the sequence identity is to SEQ ID NO:3. Preferably, the bacterialstrain for use in the invention has the 16s rRNA sequence represented bySEQ ID NO:3. In certain embodiments, the bacterial strain for use in theinvention has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%,99%, 99.5% or 99.9% identical to the 16s rRNA sequence of a bacterialstrain of Blautia wexlerae. Preferably, the bacterial strain for use inthe invention has a 16s rRNA sequence that is at least 95%, 96%, 97%,98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:2 or SEQ ID NO:4.Preferably, the sequence identity is to SEQ ID NO:4. Preferably, thebacterial strain for use in the invention has the 16s rRNA sequencerepresented by SEQ ID NO:4.

Bacterial strains that are biotypes of the bacterium deposited underaccession number DSM 14294 or biotypes of the bacteria deposited underaccession numbers NCIMB 42381 and NCIMB 42486 are also expected to beeffective for treating brain injury. A biotype is a closely relatedstrain that has the same or very similar physiological and biochemicalcharacteristics.

Strains that are biotypes of a bacterium deposited under accessionnumber DSM 14294, NCIMB 42381 or NCIMB 42486 and that are suitable foruse in the invention may be identified by sequencing other nucleotidesequences for a bacterium deposited under accession number DSM 14294,NCIMB 42381 or NCIMB 42486. For example, substantially the whole genomemay be sequenced and a biotype strain for use in the invention may haveat least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identityacross at least 80% of its whole genome (e.g. across at least 85%, 90%,95% or 99%, or across its whole genome). For example, in someembodiments, a biotype strain has at least 98% sequence identity acrossat least 98% of its genome or at least 99% sequence identity across 99%of its genome. Other suitable sequences for use in identifying biotypestrains may include hsp60 or repetitive sequences such as BOX, ERIC,(GTG)₅, or REP or [26]. Biotype strains may have sequences with at least95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to thecorresponding sequence of a bacterium deposited under accession numberDSM 14294, NCIMB 42381 or NCIMB 42486. In some embodiments, a biotypestrain has a sequence with at least 97%, 98%, 99%, 99.5% or 99.9%sequence identity to the corresponding sequence of the Blautiahydrogenotrophica strain deposited as DSM 14294 and comprises a 16S rRNAsequence that is at least 99% identical (e.g. at least 99.5% or at least99.9% identical) to SEQ ID NO: 5. In some embodiments, a biotype strainhas a sequence with at least 97%, 98%, 99%, 99.5% or 99.9% sequenceidentity to the corresponding sequence of the Blautia hydrogenotrophicastrain deposited as DSM 14294 and has the 16S rRNA sequence of SEQ IDNO:5.

Alternatively, strains that are biotypes of a bacterium deposited underaccession number DSM 14294, NCIMB 42381 or NCIMB 42486 and that aresuitable for use in the invention may be identified by using theaccession number DSM 14294 deposit, the accession number NCIMB 42381deposit, or the accession number NCIMB 42486 deposit, and restrictionfragment analysis and/or PCR analysis, for example by using fluorescentamplified fragment length polymorphism (FAFLP) and repetitive DNAelement (rep)-PCR fingerprinting, or protein profiling, or partial 16Sor 23s rDNA sequencing. In preferred embodiments, such techniques may beused to identify other Blautia hydrogenotrophica, Blautia stercoris orBlautia wexlerae strains.

In certain embodiments, strains that are biotypes of a bacteriumdeposited under accession number DSM 14294, NCIMB 42381 or NCIMB 42486and that are suitable for use in the invention are strains that providethe same pattern as a bacterium deposited under accession number DSM14294, NCIMB 42381 or NCIMB 42486 when analysed by amplified ribosomalDNA restriction analysis (ARDRA), for example when using Sau3AIrestriction enzyme (for exemplary methods and guidance see, forexample,[27]). Alternatively, biotype strains are identified as strainsthat have the same carbohydrate fermentation patterns as a bacteriumdeposited under accession number DSM 14294, NCIMB 42381 or NCIMB 42486.

Other Blautia strains that are useful in the compositions and methods ofthe invention, such as biotypes of a bacterium deposited under accessionnumber DSM 14294, NCIMB 42381 or NCIMB 42486, may be identified usingany appropriate method or strategy, including the assays described inthe examples. For instance, strains for use in the invention may beidentified by culturing bacteria and administering to mice to test inthe occlusion assay. In particular, bacterial strains that have similargrowth patterns, metabolic type and/or surface antigens to a bacteriumdeposited under accession number DSM 14294, NCIMB 42381 or NCIMB 42486may be useful in the invention. A useful strain will have comparablemicrobiota modulatory activity to the DSM 14294, NCIMB 42381 or NCIMB42486 strain. In particular, a biotype strain will elicit comparableeffects on brain injury to the effects shown in the Examples, which maybe identified by using the culturing and administration protocolsdescribed in the Examples.

A particularly preferred strain of the invention is the Blautiahydrogenotrophica strain deposited under accession number DSM 14294.This is the exemplary BH strain tested in the examples and shown to beeffective for treating disease. Therefore, the invention provides acell, such as an isolated cell, of the Blautia hydrogenotrophica straindeposited under accession number DSM 14294, or a derivative thereof, foruse in therapy, in particular for the diseases described herein.

A derivative of the strain deposited under accession number DSM 14294,NCIMB 42381 or NCIMB 42486 may be a daughter strain (progeny) or astrain cultured (subcloned) from the original. A derivative of a strainof the invention may be modified, for example at the genetic level,without ablating the biological activity. In particular, a derivativestrain of the invention is therapeutically active. A derivative strainwill have comparable microbiota modulatory activity to the original DSM14294, NCIMB 42381 or NCIMB 42486 strain. In particular, a derivativestrain will elicit comparable effects on brain injury to the effectsshown in the Examples, which may be identified by using the culturingand administration protocols described in the Examples. A derivative ofthe DSM 14294 strain will generally be a biotype of the DSM 14294strain. A derivative of the NCIMB 42381 strain will generally be abiotype of the NCIMB 42381 strain. A derivative of the NCIMB 42486strain will generally be a biotype of the NCIMB 42486 strain.

References to cells of the Blautia hydrogenotrophica strain depositedunder accession number DSM 14294 encompass any cells that have the samesafety and therapeutic efficacy characteristics as the strains depositedunder accession number DSM 14294, and such cells are encompassed by theinvention. References to cells of the Blautia stercoris strain depositedunder accession number NCIMB 42381 encompass any cells that have thesame safety and therapeutic efficacy characteristics as the strainsdeposited under accession number NCIMB 42381, and such cells areencompassed by the invention. References to cells of the Blautiawexlerae strain deposited under accession number NCIMB 42486 encompassany cells that have the same safety and therapeutic efficacycharacteristics as the strains deposited under accession number NCIMB42486, and such cells are encompassed by the invention.

In preferred embodiments, the bacterial strains in the compositions ofthe invention are viable and capable of partially or totally colonisingthe intestine.

Therapeutic Uses

The compositions of the invention are for use in treating brain injury.The examples demonstrate that the compositions of the invention aidrecovery and improve neurological function, motor abilities and socialrecognition in a mouse occlusion model of brain injury, so they may beuseful for treating brain injuries.

In some embodiments, the brain injury is a traumatic brain injury. Insome embodiments, the brain injury is an acquired brain injury. In someembodiments, the compositions of the invention are for use in treatingbrain injury resulting from trauma. In some embodiments, thecompositions of the invention are for use in treating brain injuryresulting from a tumour. In some embodiments, the compositions of theinvention are for use in treating brain injury resulting from a stroke.In some embodiments, the compositions of the invention are for use intreating brain injury resulting from a brain haemorrhage. In someembodiments, the compositions of the invention are for use in treatingbrain injury resulting from encephalitis. In some embodiments, thecompositions of the invention are for use in treating brain injuryresulting from cerebral hypoxia. In some embodiments, the compositionsof the invention are for use in treating brain injury resulting fromcerebral anoxia.

In preferred embodiments, the compositions of the invention are for usein treating stroke. The effects shown in the examples are particularlyrelevant to the treatment of stroke. Stroke occurs when blood flow to atleast a part of the brain is interrupted. Without an adequate supply ofblood to provide oxygen and nutrients to the brain tissue and to removewaste products from the brain tissue, brain cells rapidly begin to die.The symptoms of stroke are dependent on the region of the brain which isaffected by the inadequate blood flow. Symptoms include paralysis,numbness or weakness of the muscles, loss of balance, dizziness, suddensevere headaches, speech impairment, loss of memory, loss of reasoningability, sudden confusion, vision impairment, coma or even death. Astroke is also referred to as a brain attack or a cerebrovascularaccident (CVA). The symptoms of stroke may be brief if adequate bloodflow is restored within a short period of time. However, if inadequateblood flow continues for a significant period of time, the symptoms canbe permanent.

In particularly preferred embodiments, the composition of the inventioncomprises a strain of the species Blautia hydrogenotrophica and is foruse in treating stroke.

In some embodiments, the stroke is cerebral ischemia. Cerebral ischemiaresults when there is insufficient blood flow to the tissues of thebrain to meet metabolic demand. In some embodiments, the cerebralischemia is focal cerebral ischemia, i.e. confined to a specific regionof the brain. In some embodiments the cerebral ischemia is globalcerebral ischemia, i.e. encompassing a wide area of the brain tissue.Focal cerebral ischemia commonly occurs when a cerebral vessel hasbecome blocked, either partially or completely, reducing the flow ofblood to a specific region of the brain. In some embodiments the focalcerebral ischemia is ischemic stroke. In some embodiments, the ischemicstroke is thrombotic, i.e. caused by a thrombus or blood clot, whichdevelops in a cerebral vessel and restricts or blocks blood flow. Insome embodiments the ischemic stroke is a thrombotic stroke. In someembodiments, the ischemic stroke is embolic, i.e. caused by an embolus,or an unattached mass that travels through the bloodstream and restrictsor blocks blood flow at a site distant from its point of origin. In someembodiments the ischemic stroke is an embolic stroke. Global cerebralischemia commonly occurs when blood flow to the brain as a whole isblocked or reduced. In some embodiments the global cerebral ischemia iscaused by hypoperfusion, i.e. due to shock. In some embodiments theglobal cerebral ischemia is a result of a cardiac arrest.

In some embodiments the subject diagnosed with brain injury has sufferedcerebral ischemia. In some embodiments, the subject diagnosed with braininjury has suffered focal cerebral ischemia. In some embodiments, thesubject diagnosed with brain injury has suffered an ischemic stroke. Insome embodiments, the subject diagnosed with brain injury has suffered athrombotic stroke. In some embodiments, the subject diagnosed with braininjury has suffered an embolic stroke. In some embodiments, the subjectdiagnosed with brain injury has suffered global cerebral ischemia. Insome embodiments, the subject diagnosed with brain injury has sufferedhypoperfusion. In some embodiments, the subject diagnosed with braininjury has suffered a cardiac arrest. The examples demonstrate that thecompositions of the invention may be particularly useful for aidingrecovery following such brain injuries. In particularly preferredembodiments, the composition of the invention comprises a strain of thespecies Blautia hydrogenotrophica and is for use in treating a patientthat has suffered cerebral ischemia.

In some embodiments, the compositions of the invention are for use intreating cerebral ischemia. In some embodiments, the compositions of theinvention are for use in treating focal cerebral ischemia. In someembodiments, the compositions of the invention are for use treatingischemic stroke. In some embodiments, the compositions of the inventionare for use in treating thrombotic stroke. In some embodiments, thecompositions of the invention are for use in treating embolic stroke. Insome embodiments, the compositions of the invention are for use intreating global cerebral ischemia. In some embodiments, the compositionsof the invention are for use in treating hypoperfusion.

In some embodiments, the stroke is hemorrhagic stroke. Hemorrhagicstroke is caused by bleeding into or around the brain resulting inswelling, pressure and damage to the cells and tissues of the brain.Hemorrhagic stroke is commonly a result of a weakened blood vessel thatruptures and bleeds into the surrounding brain. In some embodiments, thehemorrhagic stroke is an intracerebral hemorrhage, i.e. caused bybleeding within the brain tissue itself. In some embodiments theintracerebral hemorrhage is caused by an intraparenchymal hemorrhage. Insome embodiments the intracerebral hemorrhage is caused by anintraventricular hemorrhage. In some embodiments the hemorrhagic strokeis a subarachnoid hemorrhage i.e. bleeding that occurs outside of thebrain tissue but still within the skull. In some embodiments, thehemorrhagic stroke is a result of cerebral amyloid angiopathy. In someembodiments, the hemorrhagic stroke is a result of a brain aneurysm. Insome embodiments, the hemorrhagic stroke is a result of cerebralarteriovenous malformation (AVM).

In some embodiments the subject diagnosed with brain injury has sufferedhemorrhagic stroke. In some embodiments, the subject diagnosed withbrain injury has suffered an intracerebral hemorrhage. In someembodiments, the subject diagnosed with brain injury has suffered anintraparenchymal hemorrhage. In some embodiments, the subject diagnosedwith brain injury has suffered an intraventricular hemorrhage. In someembodiments, the subject diagnosed with brain injury has suffered asubarachnoid hemorrhage. In some embodiments, the subject diagnosed withbrain injury has suffered cerebral amyloid angiopathy. In someembodiments, the subject diagnosed with brain injury has suffered abrain aneurysm. In some embodiments, the subject diagnosed with braininjury has suffered cerebral AVM.

In some embodiments, the compositions of the invention are for use intreating hemorrhagic stroke. In some embodiments, the compositions ofthe invention are for use in treating an intracerebral hemorrhage. Insome embodiments, the compositions of the invention are for use intreating an intraparenchymal hemorrhage. In some embodiments, thecompositions of the invention are for use in treating anintraventricular hemorrhage. In some embodiments, the compositions ofthe invention are for use in treating a subarachnoid hemorrhage. In someembodiments, the compositions of the invention are for use in treating acerebral amyloid angiopathy. In some embodiments, the compositions ofthe invention are for use in treating a brain aneurysm. In someembodiments, the compositions of the invention are for use in treatingcerebral AVM.

Restoration of adequate blood flow to the brain after a period ofinterruption, though effective in alleviating the symptoms associatedwith stroke, can paradoxically result in further damage to the braintissue. During the period of interruption, the affected tissue suffersfrom a lack of oxygen and nutrients, and the sudden restoration of bloodflow can result in inflammation and oxidative damage through theinduction of oxidative stress. This is known as reperfusion injury, andis well documented not only following stroke, but also following a heartattack or other tissue damage when blood supply returns to the tissueafter a period of ischemia or lack of oxygen. In some embodiments thesubject diagnosed with brain injury has suffered from reperfusion injuryas a result of stroke. In some embodiments, the compositions of theinvention are for use in treating reperfusion injury as a result ofstroke.

A transient ischemic attack (TIA), often referred to as a mini-stroke,is a recognised warning sign for a more serious stroke. Subjects whohave suffered one or more TIAs are therefore at greater risk of stroke.In some embodiments the subject diagnosed with brain injury has suffereda TIA. In some embodiments, the compositions of the invention are foruse in treating a TIA. In some embodiments, the compositions of theinvention are for use in treating brain injury in a subject who hassuffered a TIA.

High blood pressure, high blood cholesterol, a familial history ofstroke, heart disease, diabetes, brain aneurysms, arteriovenousmalformations, sickle cell disease, vasculitis, bleeding disorders, useof nonsteroidal anti-inflammatory drugs (NSAIDs), smoking tobacco,drinking large amounts of alcohol, illegal drug use, obesity, lack ofphysical activity and an unhealthy diet are all considered to be riskfactors for stroke. In particular, lowering blood pressure has beenconclusively shown to prevent both ischemic and hemorrhagic strokes [28,29]. In some embodiments, the compositions of the invention are for usein treating brain injury in a subject who has at least one risk factorfor stroke. In some embodiments the subject has two risk factors forstroke. In some embodiments the subject has three risk factors forstroke. In some embodiments the subject has four risk factors forstroke. In some embodiments the subject has more than four risk factorsfor stroke. In some embodiments the subject has high blood pressure. Insome embodiments the subject has high blood cholesterol. In someembodiments the subject has a familial history of stroke. In someembodiments the subject has heart disease. In some embodiments thesubject has diabetes. In some embodiments the subject has a brainaneurysm. In some embodiments the subject has arteriovenousmalformations. In some embodiments the subject has vasculitis. In someembodiments the subject has sickle cell disease. In some embodiments thesubject has a bleeding disorder. In some embodiments the subject has ahistory of use of nonsteroidal anti-inflammatory drugs (NSAIDs). In someembodiments the subject smokes tobacco. In some embodiments the subjectdrinks large amounts of alcohol. In some embodiments the subject usesillegal drugs. In some embodiments the subject is obese. In someembodiments the subject is overweight. In some embodiments the subjecthas a lack of physical activity. In some embodiments the subject has anunhealthy diet.

The examples demonstrate that the compositions of the invention may beuseful for treating brain injury and aiding recovery when administeredbefore the injury event occurs. Therefore, the compositions of theinvention may be particularly useful for treating brain injury whenadministered to subjects at risk of brain injury, such as stroke. Inpreferred embodiments, the composition of the invention comprises astrain of the species Blautia hydrogenotrophica and is for use intreating a patient identified as at risk of having a stroke.

In certain embodiments, the compositions of the invention are for use inreducing the damage caused by a potential brain injury, preferably astroke. The compositions may reduce the damage caused when they areadministered before the potential brain injury occurs, in particularwhen administered to a patient identified as at risk of a brain injury.

The examples show that treatment with a composition of the inventionreduces motoric damage after stroke. In some embodiments, thecompositions of the invention treat brain injury by reducing motoricdamage. The examples show that treatment with a composition of theinvention improves motor function after stroke. In some embodiments, thecompositions of the invention treat brain injury by improving motorfunction. The examples show that treatment with a composition of theinvention improves muscle strength after stroke. In some embodiments,the compositions of the invention treat brain injury by improving musclestrength. The examples show that treatment with a composition of theinvention improves memory after stroke. In some embodiments, thecompositions of the invention treat brain injury by improving memory.The examples show that treatment with a composition of the inventionimproves social recognition after stroke. In some embodiments, thecompositions of the invention treat brain injury by improving socialrecognition. The examples demonstrate that treatment with a compositionof the invention improves neurological function after stroke. In someembodiments, the compositions of the invention treat brain injury byimproving neurological function.

As demonstrated in the examples, bacterial compositions of the inventionmay be effective for treating brain injury. In certain embodiments, thecomposition may be in the form of a bacterial culture. In someembodiments, the composition may preferably be a lyophilisate.

Treatment of brain injury may refer to, for example, an alleviation ofthe severity of symptoms. Treatment of brain injury may also refer toreducing the neurological impairments following stroke. Compositions ofthe invention for use in treating stroke may be provided to the subjectin advance of the onset of stroke, for example in a patient identifiedas being at risk of stroke. Compositions of the invention for use intreating stroke may be provided after a stroke has occurred, forexample, during recovery. Compositions of the invention for use intreating stroke may be provided during the acute phase of recovery (i.e.up to one week after stroke). Compositions of the invention for use intreating stroke may be provided during the subacute phase of recovery(i.e. from one week up to three months after stroke). Compositions ofthe invention for use in treating stroke may be provided during thechronic phase of recovery (from three months after stroke).

In certain embodiments, the compositions of the invention are for use incombination with a secondary active agent. In certain embodiments, thecompositions of the invention are for use in combination with aspirin ortissue plasminogen activator (tPA). Other secondary agents include otherantiplatelets (such as clopidogrel), anticoagulants (such as heparins,warfarin, apixaban, dabigatran, edoxaban or rivaroxaban),antihypertensives (such as diuretics, ACE inhibitors, calcium channelblockers, beta-blockers or alpha-blockers) or statins. The compositionsof the invention may improve the patient's response to the secondaryactive agent.

Although there are many mechanisms associated with the pathogenesis ofstroke, numerous studies have shown that oxidative stress, inflammationand apoptosis are key factors for its pathogenic progression [30, 31].It is understood that a rapid increase in reactive oxygen species (ROS)immediately after stroke overwhelms existing antioxidant defencescausing tissue damage. Such ROS also stimulate the release ofinflammatory cytokines and matrix metalloproteinases which increaseblood brain barrier permeability and trafficking of leukocytes.Superoxide dismutase (SOD) is an endogenous antioxidant which plays arole in the prevention of oxidative injury. Therefore, enhancement ofantioxidant activities in the brain such as those of SOD is beneficialfor alleviating neuronal damage. Furthermore, inflammatory reactionsalso aggravate brain damage during and after stroke. Oxidative stressand inflammation can result in neuronal apoptosis, which can beresponsible for neuronal cell death after stroke.

Butyrate is a short chain fatty acid (SCFA), synthesised via thefermentation of otherwise non-digestible fiber by bacteria in the colon.Butyrate has been suggested to be neuroprotective, to reduce blood-brainbarrier permeability after stroke, to reduce oxidative stress, to haveanti-inflammatory effects, to inhibit neuronal apoptosis and to promotegeneral cell survival, tissue repair and recovery after stroke [32, 33,34, 35, 36].

In certain embodiments, the compositions of the invention improve thepyknosis and/or vacuolisation score of a tissue. In certain embodiments,the compositions of the invention improve the pyknosis and/orvacuolisation score of a tissue that has been subject to ischemia. Theexamples demonstrate that the compositions of the invention reducetissue damage as measured by pyknosis and vacuolisation. In certainembodiments, the compositions of the invention reduce the effect ofischemia on tissues. In certain embodiments, the compositions of theinvention reduce the amount of damage to tissues caused by ischemia. Incertain embodiments, the tissues damaged by ischemia are the cerebraltissues. In certain embodiments, the compositions of the inventionreduce necrosis or the number of necrotic cells. In certain embodiments,the compositions of the invention reduce apoptosis or the number ofapoptotic cells. In certain embodiments, the compositions of theinvention reduce the number of necrotic and apoptotic cells. In certainembodiments, the compositions of the invention prevent cell death bynecrosis and/or apoptosis. In certain embodiments, the compositions ofthe invention prevent cell death by necrosis and/or apoptosis caused byischemia. In certain embodiments, the compositions of the inventionimprove the recovery of the tissue damaged by ischemia. In certainembodiments, the compositions of the invention improve the speed ofclearance of necrotic cells and/or apoptotic cells. In certainembodiments, the compositions of the invention improve the efficacy ofthe clearance of necrotic cells and/or apoptotic cells. In certainembodiments, the compositions of the invention improve the replacementand/or regeneration of cells within tissues. In certain embodiments, thecompositions of the invention improve the replacement and/orregeneration of cells within tissues damaged by ischemia. In certainembodiments, the compositions of the invention improve the overallhistology of the tissue (for example upon a biopsy).

Modes of Administration

Preferably, the compositions of the invention are to be administered tothe gastrointestinal tract in order to enable delivery to and/or partialor total colonisation of the intestine with the bacterial strain of theinvention. Generally, the compositions of the invention are administeredorally, but they may be administered rectally, intranasally, or viabuccal or sublingual routes.

In certain embodiments, the compositions of the invention may beadministered as a foam, as a spray or a gel.

In certain embodiments, the compositions of the invention may beadministered as a suppository, such as a rectal suppository, for examplein the form of a theobroma oil (cocoa butter), synthetic hard fat (e.g.suppocire, witepsol), glycero-gelatin, polyethylene glycol, or soapglycerin composition.

In certain embodiments, the composition of the invention is administeredto the gastrointestinal tract via a tube, such as a nasogastric tube,orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneousendoscopic gastrostomy (PEG), or a port, such as a chest wall port thatprovides access to the stomach, jejunum and other suitable access ports.

The compositions of the invention may be administered once, or they maybe administered sequentially as part of a treatment regimen. In certainembodiments, the compositions of the invention are to be administereddaily. The examples demonstrate that administration provides successfulcolonisation and clinical benefits in treatment of brain injury.

In certain embodiments, the compositions of the invention areadministered regularly, such as daily, every two days, or weekly, for anextended period of time, such as for at least one week, two weeks, onemonth, two months, six months, or one year. The examples demonstratethat BH administration may not result in permanent colonisation of theintestines, so regular administration for extended periods of time mayprovide greater therapeutic benefits.

In some embodiments the compositions of the invention are administeredfor 7 days, 14 days, 16 days, 21 days or 28 days or no more than 7 days,14 days, 16 days, 21 days or 28 days. For example, in some embodimentsthe compositions of the invention are administered for 16 days. In someembodiments, the compositions of the invention are administered for 1,2, 3, 4, 5 or 6 months, over 6 months, or over 1 year.

In certain embodiments of the invention, treatment according to theinvention is accompanied by assessment of the patient's gut microbiota.Treatment may be repeated if delivery of and/or partial or totalcolonisation with the strain of the invention is not achieved such thatefficacy is not observed, or treatment may be ceased if delivery and/orpartial or total colonisation is successful and efficacy is observed.

In certain embodiments, the composition of the invention may beadministered to a pregnant animal, for example a mammal such as a humanin order to treat brain injuries developing in her child in utero and/orafter it is born.

The compositions of the invention may be administered to a patient thathas been diagnosed with brain injury or a disease or conditionassociated with brain injury, or that has been identified as being atrisk of brain injury.

The compositions of the invention may be administered to a patient thathas been identified as having an abnormal gut microbiota. For example,the patient may have reduced or absent colonisation by Blautia, and inparticular Blautia hydrogenotrophica, Blautia stercoris or Blautiawexlerae.

The compositions of the invention may be administered as a food product,such as a nutritional supplement.

Generally, the compositions of the invention are for the treatment ofhumans, although they may be used to treat animals including monogastricmammals such as poultry, pigs, cats, dogs, horses or rabbits. Thecompositions of the invention may be useful for enhancing the growth andperformance of animals. If administered to animals, oral gavage may beused.

In some embodiments, the subject to whom the composition is to beadministered is an adult human. In some embodiments, the subject to whomthe composition is to be administered is an infant human.

Compositions

Generally, the composition of the invention comprises bacteria. Inpreferred embodiments of the invention, the composition is formulated infreeze-dried form. For example, the composition of the invention maycomprise granules or gelatin capsules, for example hard gelatincapsules, comprising a bacterial strain of the invention.

Preferably, the composition of the invention comprises lyophilisedbacteria. Lyophilisation of bacteria is a well-established procedure andrelevant guidance is available in, for example, references [37-39].Lyophilisate compositions may be particularly effective. In preferredembodiments, the composition of the invention comprises lyophilisedbacteria and is for the treatment of brain injury.

Alternatively, the composition of the invention may comprise a live,active bacterial culture. In some embodiments, the bacterial strain inthe composition of the invention has not been inactivated, for example,has not been heat-inactivated. In some embodiments, the bacterial strainin the composition of the invention has not been killed, for example,has not been heat-killed. In some embodiments, the bacterial strain inthe composition of the invention has not been attenuated, for example,has not been heat-attenuated. For example, in some embodiments, thebacterial strain in the composition of the invention has not beenkilled, inactivated and/or attenuated. For example, in some embodiments,the bacterial strain in the composition of the invention is live. Forexample, in some embodiments, the bacterial strain in the composition ofthe invention is viable. For example, in some embodiments, the bacterialstrain in the composition of the invention is capable of partially ortotally colonising the intestine. For example, in some embodiments, thebacterial strain in the composition of the invention is viable andcapable of partially or totally colonising the intestine.

In some embodiments, the composition comprises a mixture of livebacterial strains and bacterial strains that have been killed.

In preferred embodiments, the composition of the invention isencapsulated to enable delivery of the bacterial strain to theintestine. Encapsulation protects the composition from degradation untildelivery at the target location through, for example, rupturing withchemical or physical stimuli such as pressure, enzymatic activity, orphysical disintegration, which may be triggered by changes in pH. Anyappropriate encapsulation method may be used. Exemplary encapsulationtechniques include entrapment within a porous matrix, attachment oradsorption on solid carrier surfaces, self-aggregation by flocculationor with cross-linking agents, and mechanical containment behind amicroporous membrane or a microcapsule. Guidance on encapsulation thatmay be useful for preparing compositions of the invention is availablein, for example, references [40-41].

The composition may be administered orally and may be in the form of atablet, capsule or powder. Encapsulated products are preferred becauseBlautia are anaerobes. Other ingredients (such as vitamin C, forexample), may be included as oxygen scavengers and prebiotic substratesto improve the delivery and/or partial or total colonisation andsurvival in vivo. Alternatively, the probiotic composition of theinvention may be administered orally as a food or nutritional product,such as milk or whey based fermented dairy product, or as apharmaceutical product.

The composition may be formulated as a probiotic.

A composition of the invention includes a therapeutically effectiveamount of a bacterial strain of the invention. A therapeuticallyeffective amount of a bacterial strain is sufficient to exert abeneficial effect upon a patient. A therapeutically effective amount ofa bacterial strain may be sufficient to result in delivery to and/orpartial or total colonisation of the patient's intestine.

A suitable daily dose of the bacteria, for example for an adult human,may be from about 1×10³ to about 1×10¹¹ colony forming units (CFU); forexample, from about 1×10⁷ to about 1×10¹⁰ CFU; in another example fromabout 1×10⁶ to about 1×10¹⁰ CFU; in another example from about 1×10⁷ toabout 1×10¹¹ CFU; in another example from about 1×10⁸ to about 1×10¹⁰CFU; in another example from about 1×10⁸ to about 1×10¹¹ CFU.

In certain embodiments, the dose of the bacteria is at least 10⁹ cellsper day, such as at least 10¹⁰, at least 10¹¹, or at least 10¹² cellsper day.

In certain embodiments, the composition contains the bacterial strain inan amount of from about 1×10⁶ to about 1×10¹¹ CFU/g, respect to theweight of the composition; for example, from about 1×10⁸ to about 1×10¹⁰CFU/g. The dose may be, for example, 1 g, 3 g, 5 g, and 10 g.

Typically, a probiotic, such as the composition of the invention, isoptionally combined with at least one suitable prebiotic compound. Aprebiotic compound is usually a non-digestible carbohydrate such as anoligo- or polysaccharide, or a sugar alcohol, which is not degraded orabsorbed in the upper digestive tract. Known prebiotics includecommercial products such as inulin and transgalacto-oligosaccharides.

In certain embodiments, the probiotic composition of the presentinvention includes a prebiotic compound in an amount of from about 1 toabout 30% by weight, respect to the total weight composition, (e.g. from5 to 20% by weight). Carbohydrates may be selected from the groupconsisting of: fructo-oligosaccharides (or FOS), short-chainfructo-oligosaccharides, inulin, isomalt-oligosaccharides, pectins,xylo-oligosaccharides (or XOS), chitosan-oligosaccharides (or COS),beta-glucans, arable gum modified and resistant starches, polydextrose,D-tagatose, acacia fibers, carob, oats, and citrus fibers. In oneaspect, the prebiotics are the short-chain fructo-oligosaccharides (forsimplicity shown herein below as FOSs-c.c); said FOSs-c.c. are notdigestible carbohydrates, generally obtained by the conversion of thebeet sugar and including a saccharose molecule to which three glucosemolecules are bonded.

The compositions of the invention may comprise pharmaceuticallyacceptable excipients or carriers. Examples of such suitable excipientsmay be found in the reference [42]. Acceptable carriers or diluents fortherapeutic use are well known in the pharmaceutical art and aredescribed, for example, in reference [43]. Examples of suitable carriersinclude lactose, starch, glucose, methyl cellulose, magnesium stearate,mannitol, sorbitol and the like. Examples of suitable diluents includeethanol, glycerol and water. The choice of pharmaceutical carrier,excipient or diluent can be selected with regard to the intended routeof administration and standard pharmaceutical practice. Thepharmaceutical compositions may comprise as, or in addition to, thecarrier, excipient or diluent any suitable binder(s), lubricant(s),suspending agent(s), coating agent(s), solubilising agent(s). Examplesof suitable binders include starch, gelatin, natural sugars such asglucose, anhydrous lactose, free-flow lactose, beta-lactose, cornsweeteners, natural and synthetic gums, such as acacia, tragacanth orsodium alginate, carboxymethyl cellulose and polyethylene glycol.Examples of suitable lubricants include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Preservatives, stabilizers, dyes and even flavouring agentsmay be provided in the pharmaceutical composition. Examples ofpreservatives include sodium benzoate, sorbic acid, cysteine and estersof p-hydroxybenzoic acid, for example, in some embodiments thepreservative is selected from sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid. Antioxidants and suspending agents may be alsoused. A further example of a suitable carrier is saccharose. A furtherexample of a preservative is cysteine.

The compositions of the invention may be formulated as a food product.For example, a food product may provide nutritional benefit in additionto the therapeutic effect of the invention, such as in a nutritionalsupplement. Similarly, a food product may be formulated to enhance thetaste of the composition of the invention or to make the compositionmore attractive to consume by being more similar to a common food item,rather than to a pharmaceutical composition. In certain embodiments, thecomposition of the invention is formulated as a milk-based product. Theterm “milk-based product” means any liquid or semi-solid milk- orwhey-based product having a varying fat content. The milk-based productcan be, e.g., cow's milk, goat's milk, sheep's milk, skimmed milk, wholemilk, milk recombined from powdered milk and whey without anyprocessing, or a processed product, such as yoghurt, curdled milk, curd,sour milk, sour whole milk, butter milk and other sour milk products.Another important group includes milk beverages, such as whey beverages,fermented milks, condensed milks, infant or baby milks; flavoured milks,ice cream; milk-containing food such as sweets.

In some embodiments, the compositions of the invention comprise one ormore bacterial strains of the genus Blautia and do not contain bacteriafrom any other genus, or which comprise only de minimis or biologicallyirrelevant amounts of bacteria from another genus.

In certain embodiments, the compositions of the invention contain asingle bacterial strain or species and do not contain any otherbacterial strains or species. Such compositions may comprise only deminimis or biologically irrelevant amounts of other bacterial strains orspecies. Such compositions may be a culture that is substantially freefrom other species of organism. In some embodiments, such compositionsmay be a lyophilisate that is substantially free from other species oforganism.

In certain embodiments, the compositions of the invention comprise oneor more bacterial strains of the genus Blautia, for example, a Blautiahydrogenotrophica, and do not contain any other bacterial genus, orwhich comprise only de minimis or biologically irrelevant amounts ofbacteria from another genus. In certain embodiments, the compositions ofthe invention comprise a single species of Blautia, for example, aBlautia hydrogenotrophica, and do not contain any other bacterialspecies, or which comprise only de minimis or biologically irrelevantamounts of bacteria from another species. In certain embodiments, thecompositions of the invention comprise a single strain of Blautia, forexample, of Blautia hydrogenotrophica, and do not contain any otherbacterial strains or species, or which comprise only de minimis orbiologically irrelevant amounts of bacteria from another strain orspecies.

In some embodiments, the compositions of the invention comprise morethan one bacterial strain or species. For example, in some embodiments,the compositions of the invention comprise more than one strain fromwithin the same species (e.g. more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40 or 45 strains), and, optionally, do not containbacteria from any other species. In some embodiments, the compositionsof the invention comprise less than 50 strains from within the samespecies (e.g. less than 45, 40, 35, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6,5, 4 or 3 strains), and, optionally, do not contain bacteria from anyother species. In some embodiments, the compositions of the inventioncomprise 1-40, 1-30, 1-20, 1-19, 1-18, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6,1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15,16-25, or 31-50 strains from within the same species and, optionally, donot contain bacteria from any other species. In some embodiments, thecompositions of the invention comprise more than one species from withinthe same genus (e.g. more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15,17, 20, 23, 25, 30, 35 or 40 species), and, optionally, do not containbacteria from any other genus. In some embodiments, the compositions ofthe invention comprise less than 50 species from within the same genus(e.g. less than 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 8, 7, 6, 5, 4 or3 species), and, optionally, do not contain bacteria from any othergenus. In some embodiments, the compositions of the invention comprise1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3,1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or31-50 species from within the same genus and, optionally, do not containbacteria from any other genus. The invention comprises any combinationof the foregoing.

In some embodiments, the composition comprises a microbial consortium.For example, in some embodiments, the composition comprises the Blautiabacterial strain as part of a microbial consortium. For example, in someembodiments, the Blautia bacterial strain is present in combination withone or more (e.g. at least 2, 3, 4, 5, 10, 15 or 20) other bacterialstrains from other genera with which it can live symbiotically in vivoin the intestine. For example, in some embodiments, the compositioncomprises a bacterial strain of Blautia hydrogenotrophica in combinationwith a bacterial strain from a different genus. In some embodiments, themicrobial consortium comprises two or more bacterial strains obtainedfrom a faeces sample of a single organism, e.g. a human. In someembodiments, the microbial consortium is not found together in nature.For example, in some embodiments, the microbial consortium comprisesbacterial strains obtained from faeces samples of at least two differentorganisms. In some embodiments, the two different organisms are from thesame species, e.g. two different humans. In some embodiments, the twodifferent organisms are an infant human and an adult human. In someembodiments, the two different organisms are a human and a non-humanmammal.

In some embodiments, the composition of the invention additionallycomprises a bacterial strain that has the same safety and therapeuticefficacy characteristics as the Blautia hydrogenotrophica straindeposited under accession number DSM 14294, but which is not the Blautiahydrogenotrophica strain deposited under accession number DSM 14294, orwhich is not a Blautia hydrogenotrophica or which is not a Blautia.

In some embodiments in which the composition of the invention comprisesmore than one bacterial strain, species or genus, the individualbacterial strains, species or genera may be for separate, simultaneousor sequential administration. For example, the composition may compriseall of the more than one bacterial strain, species or genera, or thebacterial strains, species or genera may be stored separately and beadministered separately, simultaneously or sequentially. In someembodiments, the more than one bacterial strains, species or genera arestored separately but are mixed together prior to use.

In some embodiments, the bacterial strain for use in the invention isobtained from human adult faeces. In some embodiments in which thecomposition of the invention comprises more than one bacterial strain,all of the bacterial strains are obtained from human adult faeces or ifother bacterial strains are present they are present only in de minimisamounts. The bacteria may have been cultured subsequent to beingobtained from the human adult faeces and being used in a composition ofthe invention.

In some embodiments, the one or more Blautia bacterial strains is/arethe only therapeutically active agent(s) in a composition of theinvention. In some embodiments, the bacterial strain(s) in thecomposition is/are the only therapeutically active agent(s) in acomposition of the invention.

The compositions for use in accordance with the invention may or may notrequire marketing approval.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein said bacterial strain is lyophilised. In certainembodiments, the invention provides the above pharmaceuticalcomposition, wherein said bacterial strain is spray dried. In certainembodiments, the invention provides the above pharmaceuticalcomposition, wherein the bacterial strain is lyophilised or spray driedand wherein it is live. In certain embodiments, the invention providesthe above pharmaceutical composition, wherein the bacterial strain islyophilised or spray dried and wherein it is viable. In certainembodiments, the invention provides the above pharmaceuticalcomposition, wherein the bacterial strain is lyophilised or spray driedand wherein it is capable of partially or totally colonising theintestine. In certain embodiments, the invention provides the abovepharmaceutical composition, wherein the bacterial strain is lyophilisedor spray dried and wherein it is viable and capable of partially ortotally colonising the intestine.

In some cases, the lyophilised or spray dried bacterial strain isreconstituted prior to administration. In some cases, the reconstitutionis by use of a diluent described herein.

The compositions of the invention can comprise pharmaceuticallyacceptable excipients, diluents or carriers.

In certain embodiments, the invention provides a pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in need thereof; and wherein the disorder isbrain injury.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the amount of the bacterial strain is from about1×10³ to about 1×10¹¹ colony forming units per gram with respect to aweight of the composition.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the composition is administered at a dose of 1 g, 3g, 5 g or 10 g.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the composition is administered by a methodselected from the group consisting of oral, rectal, subcutaneous, nasal,buccal, and sublingual.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a carrier selected from the group consisting oflactose, starch, glucose, methyl cellulose, magnesium stearate, mannitoland sorbitol.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a diluent selected from the group consisting ofethanol, glycerol and water.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising an excipient selected from the group consistingof starch, gelatin, glucose, anhydrous lactose, free-flow lactose,beta-lactose, corn sweetener, acacia, tragacanth, sodium alginate,carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodiumstearate, magnesium stearate, sodium benzoate, sodium acetate and sodiumchloride.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, further comprising at least one of a preservative, anantioxidant and a stabilizer.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a preservative selected from the groupconsisting of sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein said bacterial strain is lyophilised.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein when the composition is stored in a sealedcontainer at about 4.0 or about 25.0 and the container is placed in anatmosphere having 50% relative humidity, at least 80% of the bacterialstrain as measured in colony forming units, remains after a period of atleast about: 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years,2.5 years or 3 years.

In some embodiments, the composition of the invention is provided in asealed container comprising a composition as described herein. In someembodiments, the sealed container is a sachet or bottle. In someembodiments, the composition of the invention is provided in a syringecomprising a composition as described herein.

The composition of the present invention may, in some embodiments, beprovided as a pharmaceutical formulation. For example, the compositionmay be provided as a tablet or capsule. In some embodiments, the capsuleis a gelatine capsule (“gel-cap”).

In some embodiments, the compositions of the invention are administeredorally. Oral administration may involve swallowing, so that the compoundenters the gastrointestinal tract, and/or buccal, lingual, or sublingualadministration by which the compound enters the blood stream directlyfrom the mouth.

Pharmaceutical formulations suitable for oral administration includesolid plugs, solid microparticulates, semi-solid and liquid (includingmultiple phases or dispersed systems) such as tablets; soft or hardcapsules containing multi- or nano-particulates, liquids (e.g. aqueoussolutions), emulsions or powders; lozenges (including liquid-filled);chews; gels; fast dispersing dosage forms; films; ovules; sprays; andbuccal/mucoadhesive patches.

In some embodiments the pharmaceutical formulation is an entericformulation, i.e. a gastro-resistant formulation (for example, resistantto gastric pH) that is suitable for delivery of the composition of theinvention to the intestine by oral administration. Enteric formulationsmay be particularly useful when the bacteria or another component of thecomposition is acid-sensitive, e.g. prone to degradation under gastricconditions.

In some embodiments, the enteric formulation comprises an entericcoating. In some embodiments, the formulation is an enteric-coateddosage form. For example, the formulation may be an enteric-coatedtablet or an enteric-coated capsule, or the like. The enteric coatingmay be a conventional enteric coating, for example, a conventionalcoating for a tablet, capsule, or the like for oral delivery. Theformulation may comprise a film coating, for example, a thin film layerof an enteric polymer, e.g. an acid-insoluble polymer.

In some embodiments, the enteric formulation is intrinsically enteric,for example, gastro-resistant without the need for an enteric coating.Thus, in some embodiments, the formulation is an enteric formulationthat does not comprise an enteric coating. In some embodiments, theformulation is a capsule made from a thermogelling material. In someembodiments, the thermogelling material is a cellulosic material, suchas methylcellulose, hydroxymethylcellulose orhydroxypropylmethylcellulose (HPMC). In some embodiments, the capsulecomprises a shell that does not contain any film forming polymer. Insome embodiments, the capsule comprises a shell and the shell compriseshydroxypropylmethylcellulose and does not comprise any film formingpolymer (e.g. see [44]). In some embodiments, the formulation is anintrinsically enteric capsule (for example, Vcaps® from Capsugel).

In some embodiments, the formulation is a soft capsule. Soft capsulesare capsules which may, owing to additions of softeners, such as, forexample, glycerol, sorbitol, maltitol and polyethylene glycols, presentin the capsule shell, have a certain elasticity and softness. Softcapsules can be produced, for example, on the basis of gelatine orstarch. Gelatine-based soft capsules are commercially available fromvarious suppliers. Depending on the method of administration, such as,for example, orally or rectally, soft capsules can have various shapes,they can be, for example, round, oval, oblong or torpedo-shaped. Softcapsules can be produced by conventional processes, such as, forexample, by the Scherer process, the Accogel process or the droplet orblowing process.

Culturing Methods

The bacterial strains for use in the present invention can be culturedusing standard microbiology techniques as detailed in, for example,references [45-47].

The solid or liquid medium used for culture may for example be YCFA agaror YCFA medium. YCFA medium may include (per 100 ml, approximatevalues): Casitone (1.0 g), yeast extract (0.25 g), NaHCO₃(0.4 g),cysteine (0.1 g), K₂HPO₄ (0.045 g), KH₂PO₄ (0.045 g), NaCl (0.09 g),(NH₄)₂SO₄ (0.09 g), MgSO₄.7H₂O (0.009 g), CaCl₂ (0.009 g), resazurin(0.1 mg), hemin (1 mg), biotin (1 μg), cobalamin (1 μg), p-aminobenzoicacid (3 μg), folic acid (5 μg), and pyridoxamine (15 μg).

General

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., references[48-55], etc.

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x is optional andmeans, for example, x±10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

References to a percentage sequence identity between two nucleotidesequences means that, when aligned, that percentage of nucleotides arethe same in comparing the two sequences. This alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example those described in section 7.7.18 of ref.[56]. A preferred alignment is determined by the Smith-Waterman homologysearch algorithm using an affine gap search with a gap open penalty of12 and a gap extension penalty of 2, BLOSUM matrix of 62. TheSmith-Waterman homology search algorithm is disclosed in ref. [57].

Unless specifically stated, a process or method comprising numeroussteps may comprise additional steps at the beginning or end of themethod, or may comprise additional intervening steps. Also, steps may becombined, omitted or performed in an alternative order, if appropriate.

Various embodiments of the invention are described herein. It will beappreciated that the features specified in each embodiment may becombined with other specified features, to provide further embodiments.In particular, embodiments highlighted herein as being suitable, typicalor preferred may be combined with each other (except when they aremutually exclusive).

MODES FOR CARRYING OUT THE INVENTION Example 1—Stability Testing

A composition described herein containing at least one bacterial straindescribed herein is stored in a sealed container at 25° C. or 4° C. andthe container is placed in an atmosphere having 30%, 40%, 50%, 60%, 70%,75%, 80%, 90% or 95% relative humidity. After 1 month, 2 months, 3months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years, atleast 50%, 60%, 70%, 80% or 90% of the bacterial strain shall remain asmeasured in colony forming units determined by standard protocols.

Example 2—Protective Effects on Cerebral Ischemia in Mice Summary

Five groups (1M, 2M, 3M, 4M and 5M) of 15-20 mice were tested. Onlynormally behaving animals were included in the study. The first dosingday was Day −14. The 1M and 2M groups received a PBS control solutiondaily from the first dosing day until termination. The 4M group receiveda freeze dried powder control daily from the first dosing day untiltermination. The 5M group received freeze dried bacteria daily from thefirst dosing day until termination. All treatments were given in thesecond half of the working day. The treatment order of the groups wasrandomly alternated on each study Day. On Day 1, all mice wereanesthetized. A midline incision was created in the ventral side of theneck to expose the right and left common carotid-arteries. A cerebralischemia-reperfusion FR model was then induced in the 2M, 3M, 4M and 5Mgroups by bilateral common carotid artery occlusion (BCCAO) usingvascular clips for 15 minutes. At the end of each occlusion, the clipswere removed. The 1M group, the sham-operated control group, underwentthe same surgical procedure, but without carotid arteries occlusion. The3M group received an injection of MK-801 as a positive controlimmediately after surgery. In order to keep the treatment as similar aspossible, the 1M, 2M, 4M and 5M groups were also injected with sterilesaline immediately after surgery. Half of each group were terminated atDay 15 and the other half were terminated at Day 29.

Strain

Blautia hydrogenotrophica bacterium deposited under accession number DSM14294.

Administration Schedule

Group No. of Dose Level Dose Volumes Route of No. Animals Treatment(mg/kg) (ml/kg or ml/animal) Administration 1M 15 PBS (sham n/a 10 POoperated control) 2M 20 PBS n/a 10 (negative control) 3M 20 MK-801 3mg/kg 10 IP (positive control) (immediately after surgery) 4M 20Freeze-dried 7.8 mg in 100 100 μl per animal PO Powder μl 5M 20Freeze-dried 15.6 mg in 100 μl per animal Bacteria 100 μl (bacteria dose= 2 × 10⁸)

Study Design

Days −14 to 14: Daily dose of PBS control (1M and 2M groups), freezedried powder control (4M group) or freeze dried bacteria (5M group).

Day 1: Cerebral ischemia-reperfusion FR model induced in the 2M, 3M, 4Mand 5M groups by surgery; the 1M group (sham operated control) wasoperated on, but without carotid arteries occlusion. The 3M groupreceived MK-801, and the 1M, 2M, 3M and 5M groups received sterilesaline, immediately after surgery.

Day 15: Half of the mice in each group were terminated.

Day 15 to 28: Daily dose of PBS control (1M and 2M groups), freeze driedpowder control (4M group) or freeze dried bacteria (5M group) for theremaining mice in each group.

Day 29: Termination of remaining mice.

Other Study Variables

Mortality and morbidity of all mice were checked twice a day.

Body weight measurements of all mice were taken twice a week.

Blood samples (100 μL each) were collected during 2-3 hours in arandomized order in lithium-heparin tubes from each mouse from beforetreatment up to Day −14. On Day 15, 150 μL blood samples were taken fromall mice, half the amount was taken into Li-heparin tubes and the otherhalf into EDTA tubes. Following bleeding half of the mice wereterminated. On Day 29 the same bleeding procedure was applied to thesecond half of the mice.

Faecal pellets were also collected at three time points: Day −14, Day 15and Day 29. Each take was carried out in a sterile environment (fullyaseptic=cleaned between animals), with every mouse being taken out ofthe cage and placed separately into a new sterile box for individualpellet harvesting. As many pellets as possible were collected in orderto reach a minimum of 80 mg and preferably 100 mg of material per mouse.

Inverted Screen Test

An Inverted Screen test was performed on Day −2/−1 (baseline), Day 14and Day 28. In an exemplary Inverted Screen test ([58], [59]), untrainedmice are placed individually on top of a wire mesh screen which ismounted horizontally on a metal rod above a padded surface. The rod isthen rotated 180° so that the mice are on the bottom of the screen. Thetime taken for the mouse to fall from the bottom of the screen onto thepadded surface is recorded.

An Inverted Screen test can measure time taken in seconds, or it can usethe neurological deficit score: falling between 1-10 sec=1; fallingbetween 11-25 sec=2; falling between 26-60 sec=3; falling between 61-90sec=4; falling after 90 sec=5.

Social Recognition Test

A Social Recognition test was performed on Day −2/−1 (baseline), Day 14and Day 28. In an exemplary Social Recognition Test (60), each testmouse is individually placed in a cage which is a similar size to theirhome cage and allowed to acclimatize to the cage for approximately 30minutes. A second, target mouse is then placed in the cage with the testmouse. The total duration of social investigation, defined as sniffingand close following (<2 cm apart from the tail base) of the target mouseare recorded over a period of 2 minutes. The time spent investigatingthe target mouse in the first test is denoted T1. The test is thenrepeated an hour later using the same target mouse. The time spentinvestigating the target mouse in the second test is denoted T2. A testmouse with a T2 that is less than its T1 is demonstrating socialrecognition (i.e. recognition of a familiar target mouse in the repeattest leads to less investigation and a T2 that is lower than T1).

Results

Results of the Inverted Screen test are shown in FIG. 1. The y-axis ofFIG. 1 represents the baseline value in seconds minus the later value inseconds (Day 14 or 28 depending on the x-axis). The y-axis is thereforerepresenting the change in time before falling, relative to baseline. Asthe y-axis is subtracting the later value from the baseline value, apositive y-axis value is representative of a reduction in time beforefalling (i.e. poorer performance in the test) and a negative y-axisvalue is representative of an increase in time before falling (i.e. animproved performance in the test). The treated group (5M) showed clearimprovement relative to the 2M vehicle control group, demonstrating thatthe composition of the invention aided recovering and improvedneurological function and motor abilities following the induced braininjury.

Results of the Social Recognition test are shown in FIG. 2. The y-axisof FIG. 2 represents T1-T2. Therefore, the lower the value of T2 (whichdemonstrates greater social recognition) the greater the y-axis value.At Day 14 and Day 28, the treated group (5M) showed the highest socialrecognition, demonstrating that the composition of the invention aidedrecovering and improved neurological function and social recognitionfollowing the induced brain injury.

Example 3—Effects of Bacterial Lyophilisate on SCFA Production HealthyRats

The effects of chronic administration of a lyophilisate of Blautiahydrogenotrophica strain DSM 14294 on SCFA production in healthy HIMrats were studied and the results are reported in FIG. 3. Furtherdetails regarding the experiments are provided above in the descriptionsof the figure. FIG. 3 shows that administration of BH induces asignificant increase in acetate as well as in butyrate production.

Example 4—Efficacy of B. hydrogenotrophica Studied in Human MicrobiotaAssociated Rat (HMA Rat) Model Summary

Groups of 16 germ-free rats (comprising 8 rats in the control group and8 rats in the treatment group) were inoculated with the faecalmicrobiota from a human IBS subject (IBS-HMA rats). Three successiveexperiments were carried out using faecal samples from 3 different IBSpatients. Two other groups of rats (n=10) were inoculated with faecalsamples of healthy subject (n=2 subjects; 2 groups of healthy-HMA rats)as visceral sensitivity control. Thus, there were 24 IBS-microbiotaassociated rats (control), 24 IBS microbiota associated rats treatedwith Blautix and 20 healthy-microbiota associated rats. Half of theIBS-HMA rats were then administered for 28 days with compositioncomprising the bacterial strain of B. hydrogenotrophica according to theinvention while the other half animals received a control solution.

Strain

Blautia hydrogenotrophica (BH) Strain DSM 14294.

Composition and Administration

BH lyophilisate was suspended in sterile mineral solution to aconcentration of 10¹⁰ bacteria per ml. Two ml of this suspension wasadministered daily per IBS-HMA rat, by oral gavage, for a 28 daysperiod.

The control solution was the sterile mineral solution that wasadministered daily (2 ml per rat) by oral gavage to the control group ofIBS-HMA rats.

Rats

Germ-Free male Fisher rats (aged 10 weeks) were inoculated with humanfaecal microbiota from an IBS subject (IBS-HMA rats). Sixteen rats wereinoculated with the same human faecal inoculum. Three successiveexperiments were performed with faecal samples from three different IBSsubjects. Two other groups of ten rats were inoculated with faecalsample from 2 healthy subjects (normo-sensitivity control groups).

Study Design

Day −14—Inoculation of Germ-free rats with human faecal microbiota.

Days 0 to 28—Daily dose of BH lyophilisate (assay group), or controlsolution (control group) by oral gavage

Between days 14 and 22—operation to implant electrode into the abdomen(for distension assay)

Days 22-28—Adaptation of the rats to avoid stress associated withdistension test.

Day 28—distension assay and euthanasia of animals to collect the caecalsamples for sulphides and short chain fatty acid (SCFA) analysis.

Days 0, 14 and 28—Collection of faecal samples for microbial analysis:qPCR for evaluating BH population and other commensal groups ofmiccroorganisms and enumeration of functional groups of microorganismsusing selective media and strictly anaerobic method.

Results

FIG. 4 presents the results of qPCR analysis of the B. hydrogenotrophicapopulation in faecal samples from IBS-HMA rats receiving controlsolution or BH lyophilisate. A significant increase in the BH populationwas observed at the end of the administration period (D 28) in ratsreceiving the BH lyophilisate, which confirms successful delivery of BHin the colon.

FIG. 5 reports on the impact of administration of BH on the mainfermentative metabolites, short chain fatty acids, in caecal samples ofIBS-HMA rats. Administration of BH-resulted in a significant increase inacetate concentration as well as in a significant increase in butyrateconcentration (FIG. 5B).

Example 5—Histological Evaluation of the Effect of B. hydrogenotrophicaon Cerebral Tissues in the Cerebral Ischemia-Reperfusion I/R Model inMice Summary

A number of mice were subjected to the cerebral ischemia-reperfusion I/Rmodel as outlined in Example 2 above. At termination of the experiment,at either day 15 or day 29, the mice cerebral tissues were subjected tohistopathological examinations. The ischemia model causes a restrictionof blood supply to the cerebral tissues which causes a shortage ofoxygen and glucose needed for cellular metabolism and therefore to keepthe tissue alive. Therefore, this model mimics the pathology of stroke.

In order to determine whether the compositions of the invention reducedthe effect of the ischemia-reperfusion model on cerebral tissues, thehistology of the tissues were studied upon termination of theischemia-reperfusion model. The tissues were studied for pyknotic score(which assesses the extent of cell necrosis and death) and forvacuolisation (which assesses the extent of degeneration similar toapoptosis). Accordingly, these are key markers of the number of cellsthat have entered apoptosis or necrosis processes i.e. the number ofcells that are dying. This occurs following stroke and experimentalstroke, due to cell damage, following lack of blood/oxygen. The morethese parameters are elevated, the more damage is induced by the stroke.The more these parameters are decreased, the better the composition ofthe invention is at preventing damage during stroke or helping recoveryfrom stroke.

Strain

Blautia hydrogenotrophica bacterium deposited under accession number DSM14294.

Study Design and Protocol

The ischemia-reperfusion model was performed as in Example 2. The numberof animals taken to histological examination in each study group isshown below.

Group No. of Animals No. Treatment At day 15 At day 29 1M PBS (shamoperated 6 5 control) 2M PBS 9 8 (negative control) 3M MK-801 8 6(positive control) 4M Freeze-dried Powder 5 7 5M Freeze-dried Bacteria 65

Preparation of Tissues for Histological Analysis

At termination the animals were sacrificed by CO₂ asphyxiation. Wherepossible, half of the animals were sacrificed on day 15, and the otherhalf were sacrificed on day 29. Following perfusion with saline only,ileum, caecum and colon was harvested from each mouse, transferred toseparate marked sterile tubes and stored at −80° C. Brains were alsoharvested following saline perfusion. The right hemisphere of each brainwas fixed with 2.5% Paraformaldehyde and stored at 2-8° C. The lefthemisphere of each brain was stored at −80° C.

The sections of the right hemisphere of all animals per group wereloaded onto slides followed by histological examination. Tissues weretrimmed, embedded in paraffin, sectioned at approximately 5 micronsthickness and stained with Hematoxylin & Eosin (H&E) (as well as withTUNEL reagents) using standardized protocols. The slides were examinedunder a light microscope. Pictures were taken using microscope (OlympusBX60) at magnifications of ×10, ×20 and ×40. The camera used was OlympusDP-73.

Histological Evaluation Protocol

The numbers of karyopy-knosis and apoptotic cells were counted, and themean values were calculated. Neuron count was performed manually in theright hemisphere of the mice hippocampus. Ten field of X20magnifications were evaluated for the pyknosis and vacuolization scoreas follows.

Pyknosis and Vacuolization, Semi-Quantitative Scores:

1. Grade 0=no affected cells2. Grade 1=up to 10 affected cells3. Grade 2=>10 and <20 affected cells4. Grade 3=>20 and <30 affected cells5. Grade 4=>30 and <40 affected cells6. Grade 5=>40 and <50 affected cells7. Grade 6=>50 affected cells

A score was determined for each field of X20 magnification for eachmouse in a given sample group. The scores for each sample group werethen averaged to give the mean value displayed in the graphs of theresults (FIGS. 6A and 6B).

Results Pyknosis

FIG. 6A displays the semi-quantative pyknosis score after histology H&Estaining. As expected, the PBS negative control (2M) mice subjected tothe ischemia-reperfusion test showed a significant increase in thenumber of necrotic and/or dying cells compared to the sham negativecontrol (1M i.e. mice not subjected to the ischemia-reperfusion test andwhich showed a score below Grade 1). In addition, mice administered thepositive control MK-801 (3M) showed a reduced pyknosis score, indicativeof a reduced number of necrotic cells. Strikingly, mice administered thecomposition of the invention (5M) showed an even further reducedpyknosis score, which was also significantly reduced compared to the PBSnegative control group (2M) and reduced relative to mice administeredthe freeze dried powder (4M). Accordingly, mice administered thecomposition of the invention display a significant trend in reducedtissue necrosis caused by ischemia.

Vacuolisation

FIG. 6B demonstrates the semi-quantative score of vacuolisation afterhistology H&E staining. Again, as expected, the PBS negative control(2M) mice subjected to the ischemia-reperfusion test showed asignificant increase in the number of necrotic and/or dying cellscompared to the sham negative control (1M), which showed a score justabove Grade 0. Contrary to the observations for the pyknosis score,administration of the positive control (3M) did not reduce thevacuolisation score, indicating that MK-801 fails to reduce the amountof apoptosis-triggered cell death. However, mice administered thecomposition of the invention (5M) demonstrated a significant reductionin vacuolisation score, lower than all the other groups. Accordingly,the compositions of the present invention reduce the amount of apoptoticcell death caused by ischemia.

Representative Histology

FIG. 6C provides representative photographs of the H&E stained slidesfrom each of the test animals. These images were used to prepare thesemi-quantitative scores discussed above. The 5M group (administered thecomposition of the present invention) displays a greater density andnumber of cells, and reduced vacuolisation, compared to the 4M group(freeze-dried powder control) and are more similar to the MK-801positive control. Indeed, the mice administered the bacterialcomposition of the present invention shows a greatly improve tissuehistology compared to the ischemia mice model negative control (2M).

CONCLUSIONS

The B. hydrogenotrophica strain of the present invention significantlyimproves cerebral tissue histology of mice subjected to theischemia-reperfusion test compared to both of the negative controls. Inline with this, the bacterial composition reduced the number of necroticand dead cells, as well as reducing the number of cells undergoingapoptosis (vacuolisation). Although the freeze-dried powder also causeda slight reduction in pyknosis and vacuolisation scores, this reductionwas not observed to the same extent in this experimental group as thegroup administered the bacterial composition of the invention. Apossible reason for the improved histology in the lyophilised control isthat the freeze-dried powder contains sugars that could have an effecton the recovery of cells subjected to a reduced level of oxygen andmetabolites (due to the ischemia-reperfusion test). Nevertheless, thebacterial strain of the present invention shows clear improved histologyin both the pyknosis and vacuolisation scores compared to thefreeze-dried powder negative control. Accordingly, this bacterial straindemonstrates an ability to prevent cell death in cerebral tissues (bynecrosis or apoptosis) and/or aid in the recovery of the cerebral tissueafter ischemia-induced damage.

SequencesSEQ ID NO: 1 (Blautia stercoris strain GAM6-1 16S ribosomal RNA gene, partial sequence -HM626177) 1tgcaagtcga gcgaagcgct tacgacagaa ccttcggggg aagatgtaag ggactgagcg 61gcggacgggt gagtaacgcg tgggtaacct gcctcataca gggggataac agttggaaac 121ggctgctaat accgcataag cgcacggtat cgcatgatac agtgtgaaaa actccggtgg 181tatgagatgg acccgcgtct gattagctag ttggaggggt aacggcccac caaggcgacg 241atcagtagcc ggcctgagag ggtgaacggc cacattggga ctgagacacg gcccagactc 301ctacgggagg cagcagtggg gaatattgca caatggggga aaccctgatg cagcgacgcc 361gcgtgaagga agaagtatct cggtatgtaa acttctatca gcagggaaga aaatgacggt 421acctgactaa gaagccccgg ctaactacgt gccagcagcc gcggtaatac gtagggggca 481agcgttatcc ggatttactg ggtgtaaagg gagcgtagac ggaagagcaa gtctgatgtg 541aaaggctggg gcttaacccc aggactgcat tggaaactgt ttttcttgag tgccggagag 601gtaagcggaa ttcctagtgt agcggtgaaa tgcgtagata ttaggaggaa caccagtggc 661gaaggcggct tactggacgg taactgacgt tgaggctcga aagcgtgggg agcaaacagg 721attagatacc ctggtagtcc acgccgtaaa cgatgaatac taggtgttgg ggagcaaagc 781tcttcggtgc cgcagcaaac gcaataagta ttccacctgg ggagtacgtt cgcaagaatg 841aaactcaaag gaattgacgg ggacccgcac aagcggtgga gcatgtggtt taattcgaag 901caacgcgaag aaccttacca agtcttgaca tcgatctgac cggttcgtaa tggaaccttt 961ccttcgggac agagaagaca ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt 1021gggttaagtc ccgcaacgag cgcaacccct atcctcagta gccagcaggt gaagctgggc 1081actctgtgga gactgccagg gataacctgg aggaaggcgg ggacgacgtc aaatcatcat 1141gccccttatg atttgggcta cacacgtgct acaatggcgt aaacaaaggg aagcgagccc 1201gcgaggggga gcaaatccca aaaataacgt cccagttcgg actgcagtct gcaactcgac 1261tgcacgaagc tggaatcgct agtaatcgcg aatcagaatg tcgcggtgaa tacgttcccg 1321ggtcttgtac acaccgcccg tcacaccatg ggagtcagta acgcccgaag tcSEQ ID NO: 2 (Blautia wexlerae strain WAL 14507 16S ribosomal RNA gene, partial sequence -EF036467) 1caagtcgaac gggaattant ttattgaaac ttcggtcgat ttaatttaat tctagtggcg 61gacgggtgag taacgcgtgg gtaacctgcc ttatacaggg ggataacagt cagaaatggc 121tgctaatacc gcataagcgc acagagctgc atggctcagt gtgaaaaact ccggtggtat 181aagatggacc cgcgttggat tagcttgttg gtggggtaac ggcccaccaa ggcgacgatc 241catagccggc ctgagagggt gaacggccac attgggactg agacacggcc cagactccta 301cgggaggcag cagtggggaa tattgcacaa tgggggaaac cctgatgcag cgacgccgcg 361tgaaggaaga agtatctcgg tatgtaaact tctatcagca gggaagatag tgacggtacc 421tgactaagaa gccccggcta actacgtgcc agcagccgcg gtaatacgta gggggcaagc 481gttatccgga tttactgggt gtaaagggag cgtagacggt gtggcaagtc tgatgtgaaa 541ggcatgggct caacctgtgg actgcattgg aaactgtcat acttgagtgc cggaggggta 601agcggaattc ctagtgtagc ggtgaaatgc gtagatatta ggaggaacac cagtggcgaa 661ggcggcttac tggacggtaa ctgacgttga ggctcgaaag cgtggggagc aaacaggatt 721agataccctg gtagtccacg ccgtaaacga tgaataacta ggtgtcgggt ggcaaagcca 781ttcggtgccg tcgcaaacgc agtaagtatt ccacctgggg agtacgttcg caagaatgaa 841actcaaagga attgacgggg acccgcacaa gcggtggagc atgtggttta attcgaagca 901acgcgaagaa ccttaccaag tcttgacatc cgcctgaccg atccttaacc ggatctttcc 961ttcgggacag gcgagacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg 1021gttaagtccc gcaacgagcg caacccctat cctcagtagc cagcatttaa ggtgggcact 1081ctggggagac tgccagggat aacctggagg aaggcgggga tgacgtcaaa tcatcatgcc 1141ccttatgatt tgggctacac acgtgctaca atggcgtaaa caaagggaag cgagattgtg 1201agatggagca aatcccaaaa ataacgtccc agttcggact gtagtctgca acccgactac 1261acgaagctgg aatcgctagt aatcgcggat cagaatgccg cggtgaatac gttcccgggt 1321cttgtacaca ccgcccgtca caccatggga gtcagtaacg cccgaagtca gtgacctaac 1381tgcaaagaag gagctgccga aggcgggacc gatgactggg gtgaagtcgt aacaaggtSEQ ID NO: 3 (consensus 16S rRNA sequence for Blautia stercoris strain 830)TTTKGTCTGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGCTTACGACAGAACCTTCGGGGGAAGATGTAAGGGACTGAGCGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCTCATACAGGGGGATAACAGTTGGAAACGGCTGCTAATACCGCATAAGCGCACAGTATCGCATGATACAGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCCTGAGAGGGTGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCGACGCCGCGTGAAGGAAGAAGTATCTCGGTATGTAAACTTCTATCAGCAGGGAAGAAAATGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGAAGAGCAAGTCTGATGTGAAAGGCTGGGGCTTAACCCCAGGACTGCATTGGAAACTGTTTTTCTTGAGTGCCGGAGAGGTAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGACGGTAACTGACGTTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTTGGGGAGCAAAGCTCTTCGGTGCCGCAGCAAACGCAATAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTATTCGAAGCAACGCGAAGAACCTTACCAAGTCTTGACATCGATCTGACCGGTTCGTAATGGAACCTTTCCTTCGGGACAGAGAAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCGTCAGTAGCCAGCAGGTAAAGCTGGGCACTCTGAGGAGACTGCCAGGGATAACCTGGAGGAAGGCGGGGACGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTACAATGGCGTAAACAAAGGGAAGCGAGCCCGCGAGGGGGAGCAAATCCCAAAAATAACGTCCCAGTTCGGACTGCAGTCTGCAACTCGACTGCACGAAGCTGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTCAGTAACGCCCGAAGTCAGTGACCCAACCTTAGGGAGGGAGCTGCCGAAGGCGGGATTGATAACTGGGGTGAAGTCTAGGGGGTSEQ ID NO: 4 (consensus 16S rRNA sequence for Blautia wexlerae strain MRX008)TTCATTGAGACTTCGGTGGATTTAGATTCTATTTCTAGTGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCTTATACAGGGGGATAACAGTCAGAAATGGCTGCTAATACCGCATAAGCGCACAGAGCTGCATGGCTCAGTGTGAAAAACTCCGGTGGTATAAGATGGACCCGCGTTGGATTAGCTTGTTGGTGGGGTAACGGCCCACCAAGGCGACGATCCATAGCCGGCCTGAGAGGGTGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCGACGCCGCGTGAAGGAAGAAGTATCTCGGTATGTAAACTTCTATCAGCAGGGAAGATAGTGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGTGTGGCAAGTCTGATGTGAAAGGCATGGGCTCAACCTGTGGACTGCATTGGAAACTGTCATACTTGAGTGCCGGAGGGGTAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGACGGTAACTGACGTTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTCNGGGGAGCATGGCTCTTCGGTGCCGTCGCAAACGCAGTAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAGTCTTGACATCCGCCTGACCGATCCTTAACCGGATCTTTCCTTCGGGACAGGCGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCCTCAGTAGCCAGCATTTAAGGTGGGCACTCTGGGGAGACTGCCAGGGATAACCTGGAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTACAATGGCGTAAACAAAGGGAAGCGAGATCGTGAGATGGAGCAAATCCCAAAAATAACGTCCCAGTTCGGACTGTAGTCTGCAACCCGACTACACGAAGCTGGAATCGCTAGTAATCGCGGATCAGAATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTCAGTAACGCCCGAAGTCAGTGACCTAACTGCAAAGAAGGAGCTGCCGAASEQ ID NO: 5 (Blautia hydrogenotrophica strain S5a36 16S ribosomal RNA gene, partial sequence -X95624.1) 1gatgaacgct ggcggcgtgc ttaacacatg caagtcgaac gaagcgatag agaacggaga 61tttcggttga agttttctat tgactgagtg gcggacgggt gagtaacgcg tgggtaacct 121gccctataca gggggataac agttagaaat gactgctaat accgcataag cgcacagctt 181cgcatgaagc ggtgtgaaaa actgaggtgg tataggatgg acccgcgttg gattagctag 241ttggtgaggt aacggcccac caaggcgacg atccatagcc ggcctgagag ggtgaacggc 301cacattggga ctgagacacg gcccaaactc ctacgggagg cagcagtggg gaatattgca 361caatggggga aaccctgatg cagcgacgcc gcgtgaagga agaagtatct cggtatgtaa 421acttctatca gcagggaaga aagtgacggt acctgactaa gaagccccgg ctaattacgt 481gccagcagcc gcggtaatac gtaaggggca agcgttatcc ggatttactg ggtgtaaagg 541gagcgtagac ggtttggcaa gtctgatgtg aaaggcatgg gctcaacctg tggactgcat 601tggaaactgt cagacttgag tgccggagag gcaagcggaa ttcctagtgt agcggtgaaa 661tgcgtagata ttaggaggaa caccagtggc gaaggcggcc tgctggacgg taactgacgt 721tgaggctcga aagcgtgggg agcaaacagg attagatacc ctggtagtcc acgctgtaaa 781cgatgaatac taggtgtcgg gtggcaaagc cattcggtgc cgcagcaaac gcaataagta 841ttcccacctg gggagtacgt tcgcaagaat gaaactcaaa ggaattgacg gggacccgca 901caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aaatcttgac 961atccctctga ccgggaagta atgttccctt ttcttcggaa cagaggagac aggtggtgca 1021tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 1081tattcttagt agccagcagg tagagctggg cactctaggg agactgccag ggataacctg 1141gaggaaggtg gggatgacgt caaatcatca tgccccttat gatttgggct acacacgtgc 1201tacaatggcg taaacaaagg gaagcgaagg ggtgacctgg agcaaatctc aaaaataacg 1261tctcagttcg gattgtagtc tgcaactcga ctacatgaag ctggaatcgc tagtaatcgc 1321gaatcagaat gtcgcggtga atacgttccc gggtcttgta cacaccgccc gtcacaccat 1381gggagtcagt aacgcccgaa gtcagtgacc caaccnaaag gagggagctg ccgaaggtgg 1441gactgataac tggggtga

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1.-31. (canceled)
 32. A method of treating a brain injury in a subjectin need thereof, comprising administering to the subject a compositionthat comprises a therapeutically effective amount of a bacteria strainof the genus Blautia, wherein the bacteria strain comprises a 16S rRNAgene sequence having at least 95% sequence identity to thepolynucleotide sequence of SEQ ID NO:5, and wherein the administering iseffective to treat the brain injury.
 33. The method of claim 32, whereinthe administering is effective to improve a loss of neurologicalfunction, motor abilities and/or social recognition associated with thebrain injury.
 34. The method of claim 32, wherein the brain injury isresulting from a trauma, a tumor, a brain hemorrhage, an encephalitis, acerebral hypoxia, or a cerebral anoxia.
 35. The method of claim 34,wherein the brain hemorrhage is an intracerebral hemorrhage, anintraparenchymal hemorrhage, an intraventricular hemorrhage, or asubarachnoid hemorrhage.
 36. The method of claim 32, wherein the braininjury is caused by a cerebral amyloid angiopathy, a brain aneurysm, ora cerebral arteriovenous malformation (AVM).
 37. The method of claim 32,wherein the therapeutically effective amount comprises from about 1×10³to about 1×10¹¹ colony forming units (CFU).
 38. The method of claim 32,wherein the composition comprises the bacterial strain in an amount offrom about 1×10⁶ to about 1×10¹¹ colony forming units per gram (CFU/g),with respect to the weight of the composition.
 39. The method of claim32, wherein the composition comprises no more than de minimis amounts ofother bacteria strains.
 40. The method of claim 32, wherein the bacteriastrain is live.
 41. The method of claim 32, wherein the bacteria strainis capable of at least partially colonizing an intestine of the subject.42. The method of claim 32, wherein the administering comprises oral,rectal, nasal, buccal, sublingual, or subcutaneous administration. 43.The method of claim 32, wherein the composition is formulated fordelivery to an intestine of the subject.
 44. The method of claim 32,wherein the composition is encapsulated.
 45. The method of claim 32,wherein the bacteria strain is lyophilized.
 46. The method of claim 32,wherein the bacteria strain is of the species Blautia hydrogenotrophica.47. The method of claim 32, wherein the bacteria strain comprises a 16SrRNA gene sequence having at least 98% sequence identity to thepolynucleotide sequence of SEQ ID NO:5
 48. The method of claim 32,wherein the bacteria strain comprises a 16S rRNA gene sequence that isthe polynucleotide sequence of SEQ ID NO:5.
 49. The method of claim 32,wherein the bacteria strain is the strain deposited under accessionnumber DSM 14294 or a biotype of the strain deposited under accessionnumber DSM
 14294. 50. The method of claim 49, wherein the biotype is abacteria strain that has the same carbohydrate fermentation pattern asthe bacteria strain deposited under accession number DSM
 14294. 51. Themethod of claim 32, wherein the composition further comprises apharmaceutically acceptable excipient or carrier.